A family of hormones, comprising insulin, insulin-like growth factors (I and II), bombyxin, molluscan insulin-related peptide and relaxin, has been identified and designated as "insulin-related." Blundell and Humbel, 1980, Nature 287:781-787; Bullesbach and Schwabe, 1991, J. Biol. Chem. 266:10754-10761. The proteins comprising this family of hormones represents a group of polypeptides having homologous primary and secondary structure but divergent biological functions.
Relaxin has been purified from a variety of species including porcine, murine, equine, shark, tiger, rat, dogfish and human. In the human, relaxin is most abundantly found in the corpora lutea (CL) of pregnancy. Mature human relaxin is a hormonal peptide of approximately 6000 daltons which facilitates the birth process by remodelling the reproductive tract before parturition. More specifically, relaxin appears to mediate the restructuring of connective tissues in target organs to obtain the required changes in organ structure during pregnancy and parturition. See, Hisaw, 1926, Proc. Soc. Exp. Biol. Med. 23:661-663; Schwabe, et al., 1977, Biochem. Biophys. Res. Comm. 75:503-570; James, et al., 1977, Nature, 267:544-546. A concise review of relaxin was provided by Sherwood, D. in The Physiology of Reproduction, Chapter 16, "Relaxin", Knobil, E. and Neill, J., et al. (eds.), (Raven Press Ltd., New York), pp. 585-673 (1988).
While predominantly a hormone of pregnancy, relaxin has also been detected in the non-pregnant female as well as in the male. Bryant-Greenwood, 1982, Endocrine Reviews 3:62-90; Weiss, 1984, Ann. Rev. Physiol. 46:43-52.
Two human gene forms encoding for human relaxin have been identified, (H1) and (H2). Hudson, et al., 1983, Nature 301 628-631; Hudson, et al., 1984, EMBO J., 3:2333-2339; and U.S. Pat. Nos. 4,758,516 and 4,871,670. Only one of the gene forms (H2) has been found to be transcribed in CL. It remains unclear whether the (H1) form is expressed at another tissue site, or whether it represents a pseudo-gene. When synthetic human relaxin (H2) and certain human relaxin analogs were tested for biological activity, the tests revealed a relaxin core necessary for biological activity as well as certain amino acid substitutions for methionine that did not affect biological activity. Johnston, et al., in Peptides: Structure and Function, Proc. Ninth American Peptide Symposium, Deber, C. M., et al. (eds.) (Pierce Chem. Co. 1985).
Methods of making relaxin are described in U.S. Pat. No. 4,835,251 and in co-pending U.S. Pat. No. 5,464,756 (PCT US90/02085) and PCT US94/06997. Methods of using relaxin in cardiovascular therapy and in the treatment of neurodegenerative diseases are described in U.S. Pat. No. 5,166,191 and in PCT US92/06927. Certain formulations of human relaxin are described in allowed U.S. Pat. No. 5,451,572.
The structure and biological function and activity of the remaining members of the insulin-related family have been extensively studied. See, e.g. Robinson and Fritz, 1981, Biol. Reprod. 24:1032-1041; Soder, et al., 1992, Endocrinology 131:2344-2350; Luthman, et al., 1989, Eur. J. Biochem 180(2):259-65; Jhoti, et al., 1987, FEBS Lett. 219:419-425; Smit, et al., 1988, Nature 331:535-538. Among the structural features shared between relaxin and the remaining members of the insulin-related family of hormones are molecular weight, a "two-chain" structure comprising a B-chain, a connecting C-peptide, and an A-chain, and the number and disposition of disulfide links.
Despite these similarities, the proteins comprising the insulin-related family have been found to have distinct biological functions and activities. It has been reported that this distinction is in large part a consequence of differences between a few type-specific amino acid residues. For example, the difference between the glycine in position A14 of human type II relaxin and the isoleucine in the equivalent position (A10) of insulin is considered critical in distinguishing between the biological activity of the two proteins. Schwabe and Bullesbach, 1994, FASEB J. 8:1-2.
A protein having the structural characteristics of insulin, insulin-like growth factor (IGF) and relaxin has been isolated recently from Leydig cells of the testes. Burkhardt, et al., 1993, Genomics 20:13-19. This protein, designated as a Leydig cell-specific insulin-like peptide (Ley I-L), has been characterized as being "insulin-like" due to the genomic location of the gene encoding Ley I-L vis a vis the gene encoding insulin (as compared to the genomic location of the gene encoding either relaxin or IGF). Burkhardt, et al., 1993, Genomics 20:13-19.
The Ley I-L protein has been characterized also as insulin-like, rather than either IGF-like or relaxin-like, based upon the protein's C-peptide chain length. More specifically, the C-peptide length of the Ley I-L protein is 49 amino acids, as compared to the 35 amino acid length of proinsulin C-peptide, the twelve amino acid length of the known proIGF C-peptides and the over one-hundred amino acid C-peptide length of prorelaxin. Finally, Ley I-L has been designated insulin-like based on the observation that the protein is expressed exclusively in prenatal and postnatal testicular Leydig cells. Burkhardt, et al., supra.
On the basis of the protein's similarities to insulin and the source of such protein, it was reported that the Ley I-L protein is implicated in testicular function. Id., Adham, et al., 1993, J. Rio. Chem. 268(35) :26668-6672.
In consultation with the inventors of the present invention, Tashima, et al., 1995, J. Clin. Endocrinal. Metab. 80:707-710, have investigated the accuracy of previous reports providing that the Ley I-L gene was only expressed in Leydig cells. Specifically, Tashima, et al. investigated whether the Ley I-L gene was present and expressed in female reproductive tissues, the human corpus luteum, trophoblasts, fetal membranes and breast tissue. As with the case with H2 relaxin, Tashima, et al. determined that the Ley I-L protein can be found in human corpus luteum and trophoblast. Unlike H2 relaxin, however, Ley I-L was not found to be expressed in fetal membranes, decidua and breast tissue.
Neither the Burkhardt/Adham group nor the Tashima group have reported the biological function of the Ley I-L protein. Thus, while the structure of this putative Ley I-L protein has been identified, no correct activity or use was known for this protein until the present invention, which completed the discovery of RLF through the identification and proof of its utility.